Previously, we demonstrated the existence of a set of eight genes which are essential for the secretion of pertussis toxin (PT) from B. pertussis. These genes, termed ptl genes, encode transport proteins that share homology with a family of proteins involved in the transport of both DNA and proteins, across bacterial membranes. We now have evidence for a ninth secretion gene. This small gene, ptlI, is predicted to encode a protein of 6.8 kDa and is located between ptlD and ptlE. PtlI shares homology with VirB7 which is required for transport of T-DNA across the membranes of the plant pathogen Agrobacterium tumefaciens. Deletion of the ptlI region from B. pertussis has no discernable effect of PtlE but resulted in loss of detectable PtlF, suggesting that PtlI may interact with and stabilize PtlF. These results are the first evidence for interactions between Ptl proteins. This work increases our knowledge of the secretion of PT and may aid in the construction of strains of B. pertussis which efficiently produce and secrete PT. Such strains would be useful for vaccine production.